CAR activation mechanism: What is unique about this system is the fact that xenobiotics do not directly bind to CAR to activate it. We previously determined that threonine 48 of endogenous CAR is phosphorylated in mouse primary hepatocytes and that phenobarbital treatment de-phosphorylates this threonine, activating CAR and translocating it into the nucleus. We identified protein phosphatase 2A as the enzyme that de-phosphorylates threonine 48 of CAR. Moreover, receptor for activated C-kinase1 (RACK1) was characterized as the essential regulatory subunit that activates the PP2A core enzyme to de-phosphorylate threonine 48 of CAR. Phenobarbital indirectly activates nuclear receptor CAR by de-phosphorylating threonine 38. Here we have now determined the underlying mechanism through which phenobarbital activates CAR. Phenobarbital binding to EGFR initiates cell signaling to de-phosphorylate tyrosine 52 of RACK1. Non-phosphorylated RACK1, acting as the regulatory subunit, stimulates protein phosphatase 2A to de-phosphorylate threonine 38 of CAR for its activation. An inactive CAR is present as its homodimer in the cytoplasm. Upon treatment with CAR activators, CAR dissociates its homodimer and a resulting monomer exposes the RACK1/PP2c binding site for dephosphorylation and activation. EGF signal regulates this monomer -dimer conversion through phosphor-ERK1/2-CAR interactions. Our investigations have defined the phenobarbital-EGFR-RACK1/PP2Ac-CAR as the principle mechanism for CAR activation. Threonine 38 of CAR is conserved as a phosphorylation motif in the majority of human nuclear receptors such as estrogen receptors. We examined serine 212 of human estrogen receptor (serine 216 in mouse ERalpha) in various mouse tissues and cells. Utilizing phosphor-S216 peptide antibody, it is now confirmed that mouse ER phosphorylated at serine 216 is, at least,expressed in immune cells such as neutrophils and brain microglia. Phosphorylated ERalpha regulates neutrophil migration and infiltration into the uterus, that in microglia exerts an anti-inflammatory activity. Therefore, this phosphorylation motif may confer distinct functions to a given nuclear receptor far beyond that of CAR. In addition, this phosphorylation is now characterized as a conserved degradation signal in the nuclear receptor family proteins. Xenobiotic-signal crosstalk mechanism: Upon activation by xenobiotics, CAR regulates genes differently from one another, conferring specificity to CAR-regulated gene expression. CAR acquires this specificity via crosstalk with cell signaling. We have identified various endogenous cell signals as the essential regulator of CAR activation and function: p38 MAPK, SGK2 and the GADD45 (growth arrest and DNA-damage inducible 45). Given these findings, we are investigating the molecular mechanisms by which these signaling regulate CAR activation and functions. CAR-mediated diseases: Chronic treatment with drugs, such as phenobarbital, is known to activate CAR and cause hepatocellular carcinoma (HCC) in rodents. We have now characterized KCNK1 and GADD45 as a CAR target for phenobarbital promotion of HCC development: CAR interacts with GADD45 protein and this interaction inhibits phosphorylation of JNK1 and p38, thus repressing apoptosis and possibly promoting tumor genesis. Polybrominated diphenyl ether PBDE-47 (2, 2, 4, 4-tetrabromodipheyl ether), a persistent and bioaccumulative brominated flame retardant, is found in nearly all Americans. It has been associated with a wide variety of health effects in people (e.g., endocrine disruption, reproductive effects, developmental neurotoxicity)is now found to be a strong human CAR activator. Statins is now found to activate the PXR-SGK2 signaling to induce hepatic gluconeogenesis, thus providing the molecular basis for understanding an increase of blood glucose levels and a risk of developing type 2 diabetes, side effects caussed by statin treatment.